ABSTRACT / PROJECT SUMMARY Aflatoxin, produced by the fungi Aspergillus flavus and Aspergillus parasiticus, is a well-known environmental carcinogen that causes mutations and ultimately leads to hepatocellular carcinoma (HCC), one of the leading causes of cancer deaths worldwide. Chronic dietary exposure to aflatoxin B1 (AFB1), the most mutagenic aflatoxin, and chronic infections with the hepatitis B virus (HBV) or hepatitis C virus (HCV) are the two major etiological factors for the development of HCC. Nucleotide excision repair is the repair mechanism by which the bulky lesions induced by AFB1 are removed from the genome. Despite the progress in our knowledge of the AFB1-DNA adduct, the formation and removal kinetics of the bulky DNA lesion throughout the genome and the factors affecting the damage formation and repair efficiency remain elusive. Our long-term goal of this project is to better understand how the kinetics of AFB1-induced DNA damage formation and repair contributes to human hepatic mutagenesis. The overall objective of this particular proposal, which is an initial step to achieve our long-term goal, is to combine biochemistry, genetics, adductomics and computational approaches to investigate effects of histone modifications and three-dimensional (3D) genome organization on AFB1-induced DNA damage formation and repair, and to determine the correlations between AFB1-DNA adduct spectra or repair efficiencies and mutational spectra of AFB1 in human HCC. Our central hypothesis is that human hepatic mutagenesis correlates with the AFB1-induced DNA damage formation and/or repair events, which are affected by histone modifications and 3D genome organization. We propose two specific aims to test our hypothesis and accomplish the objective: 1)! Genome-wide Analysis of AFB1-induced DNA Damage Formation and Repair Kinetics as a Function of Histone Modifications and 3D Genome Organization (Aim1, K99 and R00 phase). 2) Determine the Correlations Between AFB1-DNA Adduct Spectra or Repair Efficiencies and Mutational Spectra of AFB1 in Human HCC (Aim2, R00 phase). We expect the following outcomes: Determination of the effect of histone acetylation on AFB1-DNA adducts formation and repair efficiency; determination of the effect of 3D genome organization on AFB1-DNA adducts formation and repair efficiency; identification of AFB1-induced DNA damage hot spots and repair cold spots in cancer-associated genes; determination of the correlations between AFB1-DNA adduct spectra or repair efficiencies and mutational spectra of AFB1 in human HCC. The proposed research is significant because it will give insights into development of AFB1-associated HCC, improve prevention strategies and develop better treatment for HCC.